Interest in the glycemic index of food products has significantly increased in recent years. The glycemic index is an indicator of the relative glycemic response to dietary carbohydrates in a given food product upon human digestion and allows foods to be ranked based on the rate of release and absorption of carbohydrates. In effect, the glycemic index allows identification of so-called “good carbs” (i.e., carbohydrates with relatively low glycemic indexes) and so-called “bad carbs” (i.e., carbohydrates with relatively high glycemic indexes). Carbohydrate-conscious consumers and health care providers can use the glycemic index or related values to assist in food selections.
The glycemic index ranks carbohydrates on a scale of 0 to 100 based on changes in blood sugar levels after eating. Foods with a high GI are thought to be rapidly digested and absorbed, thereby leading to marked fluctuations in blood sugar levels. Low glycemic index values are though to produce more gradual rises, and thus flattened fluctuations, in blood sugar and insulin levels due to their slower digestion and absorption by the body. Generally, low glycemic index foods are defined as having a glycemic index of 55 or less, medium glycemic index foods as having a glycemic index of 56 to 69, and high glycemic index foods as having a glycemic index of 70 or higher.
The consumption of high-glycemic index foods generally appears to result in higher and more rapid increases in blood glucose levels than the consumption of low-glycemic index foods. Rapid increases in blood glucose signal the pancreas to increase insulin secretion. High insulin levels induced by consumption of high-glycemic index foods may cause a sharp decrease in blood glucose levels (hypoglycemia) whereas consumption of low-glycemic index foods are generally thought to result in lower and more sustained increases in blood glucose and lower insulin demands. Low glycemic diets have been reported to improve both glucose and lipid levels in people with diabetes (type 1 and type 2). Low glycemic diets have also been reported to result in benefits in weight control because they help control appetite and delay hunger. Low GI diets may also reduce insulin levels and insulin resistance. Recent studies from Harvard School of Public Health report that the risks of diseases such as type 2 diabetes and coronary heart disease are strongly influenced by the glycemic index of the overall diet. The World Health Organization (WHO) and Food and Agriculture Organization of the United Nations have recommended that people in industrialized countries base their diets on low glycemic index foods in order to prevent the most common diseases of affluence, such as coronary heart disease, diabetes, and obesity. Thus, it is often recommended that consumers modify their overall diets such that the relative amount of low glycemic index foods is increased at the expense of high glycemic index foods. Glycemic index values can be used to assist consumers and health care providers in selecting foods and possibly reducing the risk of certain diseases.
The glycemic index of a given food product is usually determined in vivo by monitoring the blood glucose level of a group of human subjects (usually about 6 or more individuals) who have ingested the food product; the blood glucose response for the food product is compared to that stimulated by ingestion of a control substance of known glycemic index over a fixed period of time and the glycemic index is calculated. The in vivo glycemic index determination method is generally considered the “gold standard” in this area. In the currently accepted test protocol, measured portions of a test food containing 10 to 50 grams of carbohydrate are fed to 6 or more healthy people after an overnight fast. Blood samples, usually from finger-pricks, are taken before the food consumption (time zero) and at 15-30 minute intervals in the two hours immediately after the food consumption and analyzed for blood glucose levels. The resulting data (typically about 7 data points) are used to prepare a blood sugar response curve (i.e., blood glucose level plotted against time) for the two hour period after consumption of the test food. The area under the blood sugar curve is related to the total rise in blood glucose levels after eating the test food. A similar test is carried out, again with overnight fasting, with the same individuals consuming an equal-carbohydrate portion of glucose sugar (the reference food having, by definition, a glycemic index of 100) or white bread having a defined GI value; the two-hour blood glucose response curves are determined and the area under the curves is measured in the same manner as done for the test food. The glycemic index of the test food is calculated by dividing the area under the curve for the test food by the area under the curve for the reference food and multiplying by 100. The use of a standard food is important for reducing the confounding influence of differences in the physical and/or other characteristics of the subjects as well as to match the physical form of the test samples (i.e., standard aqueous glucose solution for beverage determinations and standard white bread for solid food determinations). Since only about 7 data points (over a two hour period) are used to generate the blood sugar curve and the curve is assumed to pass smoothly through these limited data points, errors could arise if the glucose generation is significantly increased or decreased between the actual data points taken.
The average of the glycemic indexes from all test subjects is taken as the glycemic index of the food. Of course, the accuracy of the determination depends, at least in part, on compliance of the test subjects regarding the test protocols and the validity of the assumption that the physical and/or other characteristics of the individual subjects remains essentially constant for both the test and reference food determinations.
Since such in vivo glycemic index determination methods generally require human subjects as well as being costly and time consuming, there has been considerable interest in developing in vitro test protocols. One in vitro method, based on studies by K. N. Englyst and co-workers and illustrated in FIG. 1, involved the measurement of glucose released from a test food during timed incubation at 37° C. with a mixture of digestive enzymes using a calorimetric endpoint to determine the glucose level. Englyst et al., Brit. J. Nutr., 75, 327-337 (1996). This in vitro method has more recently been modified to include a HPLC endpoint to determine the amount of glucose released. Englyst et al., Am. J. Clin. Nutr., 69, 448-454 (1999) (hereinafter referred to as the “Englyst method” or the “in vitro Englyst method”).
The Englyst method involves mincing (or otherwise crushing or breaking up) a known amount of a test food (generally to contain about 0.5 g carbohydrate). The minced samples are incubated at 37° C. for 30 minutes with mixing in 10 ml 0.05 M HCl containing pepsin (5 g/l; to effect hydrolysis of protein) and guar gum (5 g/l; to help maintain food particles in suspension throughout the analysis). After this initial incubation, the samples are buffered to pH 5.2 using 0.5 M sodium acetate. An enzyme mixture (containing specific amounts of pancreatin, amyloglucosidase, and invertase) is then added to the buffered sample and the sample placed in a shaking water bath at 37° C. (time=0). Small glass balls are included in the samples to mechanically disrupt the physical structure of the samples during the main incubation; the added guar gum helps keep the sample in suspension by stabilizing the viscosity of the samples. At exactly 20 minutes into the main incubation, an aliquot of the sample is removed and added to absolute ethanol with mixing to stop the hydrolysis; this sample is used to determine G20 (i.e., the glucose released after 20 minutes; also referred to as “rapidly available glucose” or RAG) using HPLC. The remainder of the sample is maintained in the 37° C. bath for an additional 100 minutes at which time a second aliquot of the sample is removed and added to absolute ethanol with mixing to stop the hydrolysis; this sample is used to determine G120 (i.e., the glucose released after 120 minutes) using HPLC. The remainder of the sample is then treated as illustrated in FIG. 1 by the addition of additional enzymes and then exposure to temperatures up to 100° C. to force complete hydrolysis and, thus, determine the total glucose in the sample.
Comparing foods for which the glycemic index has been measured using the in vivo test method, the G20 or rapidly available glucose values determined using the Englyst method can be correlated with the in vivo glycemic index values of known foods. Using such correlation coefficients, the Englyst method can be used to estimate glycemic index values for test foods.
The in vitro Englyst method has, however, met with considerable criticism, especially from proponents of the in vivo glycemic index method. For example, Garsetti et al., J. Am. Coll. Nutr., 24, 441-447 (2005), used the Englyst method to estimate glycemic index values (i.e., rapidly available glucose values) and then compared them with in vivo determined values for food products (i.e., cookies) having glycemic indexes in the range of about 35 to 60. As shown in FIG. 2 of Garsetti et al. (and incorporated by reference herein), a scatter plot of in vivo determined glycemic index values versus rapidly available glucose values (i.e., as determined by the Englyst method) yielded a scatter plot with an R2 value of 0.25, indicating that the in vitro method had little predictive value.
Brand-Miller et al., Eur. J. Clin. Nutr., 58, 700-701 (2004), determined the in vivo glycemic index values for commercially available foods (one breakfast cereal and two snack bars) reportedly having low glycemic index values (i.e., ≦55) as determined by the Englyst in vitro test method. According to Brand-Miller et al., the three food products had mean in vivo glycemic index values (±sem) of 75±3, 68±5, and 65±5, respectively, and thus could not be classified as low glycemic index food products. These authors concluded that assumptions that the “laborious task of in vivo testing is no longer necessary for measuring the GI values for food” are simply incorrect and “strongly advise against the use of any in vitro method for producing GI values for food labeling purposes.” Finally, the authors “urge food manufactures to undertake GI testing only with experienced laboratories using the standardized in vivo method.”
Clearly there remains a need in the art for an accurate, precise, and reproducible in vitro method for determining the glycemic index values of food products. Moreover, there remains a need for such a method which can be applied to a large variety of food products, including solid and liquid food products. The present invention provides such in vitro testing methods.